[Histonet] Formalin-fixed paraffin embedded question

[sgoebel <@t> mirnarx.com]

Wow!!  Processing by hand...I didn't think people actually did this
anymore?  I think what your problem might be is that you are not fixing
the tissue before you submerge it into alcohol for those long periods of
time.  The "splintering" artifact as you describe probably means that
your tissues are severely over dehydrated.  This is why when you float
them in the water bath for a minute or two it goes away for awhile in
the block (basically until you get to the over dehydrated tissue again).
Try fixing the samples in 10% formalin for at least overnight before you
dunk them into alcohol.
Good Luck!!

Sarah Goebel, BA, HT(ASCP)
Histotechnologist
Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Noel
Gray
Sent: Monday, February 21, 2011 2:55 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Formalin-fixed paraffin embedded question

I am having an issue with formalin-fixed, paraffin embedded tissue that
I am
sectioning. I am using a microtome to cut the tissue (mouse brain,
cerebellum and spinal cord) in 10 um sections which I will stain using
cressyl violet. Sometimes, the tissue in a block will splinter once it
hits
the blade. Usually all samples from the same animal splinter but this is
not
always the case. If I put the block into the water bath (sectioning
surface
exposed to water or not) this seems to stop the splintering for 1-200 um
worth of tissue. However, I am afraid this may bring error into the
histological analysis of my tissue.
 
I assume it has something to do with the protocol I am using to prepare
the
tissue. I guessed that maybe the dehydration, alcohol clearing, or
paraffin
infiltration are not complete, resulting in the problem I have. However,
I
looked at various FFPE protocols and each of my wash steps are longer,
which
may be the problem? I was wondering if anyone has encountered this
before,
or if anyone knows exactly what is going on with my tissue and how I can
fix
it? Thank you. 
 
Here is my protocol:
 
-Anesthetize mouse followed by a system flush of 30 ml of PBS and then
slow
perfusion of 50 ml of 4% PFA.
-Brain and spinal cord are removed as a single, in tact unit and placed
into 70% ethanol for 4 hours
-80% EtOH for 4 hours
-90% EtOH over night
-100% EtOH #1 for 4 hours
-100% EtOH #2 for 4 hours
-100% EtOH #3 over night, forebrain cerebellum and spinal cord are
separated
-Xylene wash #1 for 4 hours
-Xylene wash #2 for 4 hours
-Xylene wash #3 over night
-Moulton paraffin wash #1 for 4 hours
-Moulton paraffin wash #2 for 4 hours
-Moulton paraffin wash #3 over night
-Tissue is embedded and then sectioned into 10 um sections
 
Again thank you for your time.
 
Noel W Gray
Neuroscience Graduate Program
SUNY Upstate Medical University
3219 Weiskotten Hall
766 Irving Ave
Syracuse, NY 13210-1630
(315) 464-8144
grayn <@t> upstate.edu
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